We are submitting this grant application in response to the NIH announcement of the Availability of Recovery Act Funds for Competitive Revision Applications as a supplement to the parent EUREKA Grant Award entitled, "Shotgun Glycomics: Linking Glycan Structure and Function." Shotgun Glycomics is a nanoscale approach that allows us to 1) fluorescently tag all free glycans released from cell or tissue glycoconjugates;2) purify the glycans;and 3) create tagged glycan libraries (TGLs) that represent the glycome of the cell or tissue. Since glycans probably function as surfaces for effector molecules to bind, the tagged glycans are printed as glycan microarrays and interrogated for recognition by biologically relevant glycan-binding proteins (GBPs) or organisms. Sequencing oligosaccharides is extremely difficult and only done by highly specialized researchers with highly sophisticated equipment. Unlike nucleic acids and proteins, glycans cannot be amplified. Thus knowing sequences is of limited value. Shotgun Glycomics allows us to obtain an archival library of glycan structures on a microarray accessible to interrogation to gain knowledge about function so that we focus our sequencing and synthetic efforts on relevant glycans. As we developed TGLs and began interrogating them with GBPs, we recognized that massive amounts of structural information were available from the binding of GBPs with known specificity to unknown glycan structures. The exquisite specificity of plant lectins, for example, has been known and documented for decades. Thus, analyzing hundreds of isolated glycans on a microarray with a specific lectin could produce metadata for each glycan, and repeating the assay with additional lectins with different specificities rapidly increased the amount of metadata. We reasoned that if we included other parameters, we should be able to define glycan structures with simple microarray analyses. Thus, we are requesting support to develop a novel approach to glycan structure that we have termed "Metadata Analysis for Glycan Structure". This method only requires equipment common to molecular biology and proteomic labs, and we will develop computational tools and algorithms to convert metadata on glycan-protein binding to glycan structure. PUBLIC HEALTH RELEVANCE: Unlike nucleic acids and proteins, glycans cannot be amplified. Thus, knowing glycan sequences is of limited value. Shotgun Glycomics provides archival libraries of glycan structures for functional analysis. However, the same glycan array can be used to obtain structural data on all of the unknown glycan using "Metadata Analysis for Glycan Structure," a novel approach to glycan structure using microarrayed glycans, glycan specific proteins, and computational tools for metadata interpretation.